Circular RNA circCHSY1 silencing inhibits the malignant progression of esophageal squamous cell carcinoma

Background CircRNAs play a crucial role in the regulation of various cancers. This study aims to investigate the involvement of circCHSY1 in the development of esophageal squamous cell carcinoma (ESCC). Methods RNA levels were quantified using qRT-PCR, and protein levels were measured by western blot. The stability of circCHSY1 was analyzed using RNase R. The functional effect of circCHSY1 on cell behavior was evaluated by CCK-8, EdU, flow cytometry, transwell, tube formation, and xenograft tumor model assays. The associations among circCHSY1, miR-1229-3p, and Tectonic-1 (TCTN1) were certified by bioinformatics analysis, dual-luciferase reporter assay, and RNA pull-down assay. Results CircCHSY1 was up-regulated in both ESCC tissues and cell lines in comparison with the control groups. Knockdown of circCHSY1 inhibited the proliferation, migration, invasion, and tube formation and promoted apoptosis of ESCC cells. Mechanistically, circCHSY1 targeted miR-1229-3p, which was downregulated in ESCC tissues and cells. Inhibition of miR-1229-3p attenuated the effects mediated by circCHSY1 suppression. Besides, miR-1229-3p bound to TCTN1, and TCTN1 overexpression restored miR-1229-3p-induced effects in ESCC cells. Animal experiments revealed that circCHSY1 silencing suppressed tumor tumorigenesis in vivo. Conclusion CircCHSY1 contributed to ESCC cell malignancy, and the underlying mechanism involved the circCHSY1/miR-1229-3p/TCTN1 axis, providing potential therapeutic targets for ESCC.


Introduction
More than 450,000 new cases are diagnosed with esophageal cancer every year, placing it ninth among all cancer types [1,2].Pathologically, esophageal squamous cell carcinoma (ESCC) accounts for over 90% of these cancers [3].Although current treatment methods, such as drug therapy and targeted therapy, can significantly improve the therapeutic effect for ESCC patients, the prognosis remains poor, especially in the advanced stage [4,5].Therefore, it is necessary to investigate the molecular pathogenesis of ESCC and identify molecular markers and therapeutic targets for the disease.
Haiquan He and Ying Chen are contribute equally to this study.
MicroRNAs (miRNAs) are non-coding small molecules that play a crucial role in tumorigenesis.Abnormal expression of certain miRNAs has been detected in ESCC tissues, contributing to the development and progression of the disease.For instance, miR-375 is highly expressed in ESCC, promoting tumor cell growth and metastasis [14].Moreover, miR-34a inhibited ESCC cell proliferation and migration [15].Another miRNA, miR-1229-3p, was found to promote the progression of some cancers such as gastric cancer [16] and breast cancer [17].However, the role of miR-1229-3p in ESCC progression remains unclear.
Herein, we analyzed circCHSY1 and miR-1229-3p expression, and determined the role of circCHSY1 in ESCC cell malignancy.Additionally, the present work investigated whether the regulation of circCHSY1 in ESCC progression involved miR-1229-3p.

Human samples
The study was carried out with the approval of the Ethics Committee of Gaozhou People's Hospital and was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki 56 pairs of fresh ESCC tissues and adjacent normal tissues were obtained from this hospital.Informed consent was obtained from all participants before sample collection.The collected tissue specimens were stored at − 80 °C.

qRT-PCR
In line with the guidebook of GoldenstarTM RT6 cDNA Synthesis Kit (Chinese Indigo), the isolated RNA by TRIzol ™ was used to synthesize complementary DNA.Premix Ex Taq ™ II (Chinese Indigo) was used for real-time PCR.RNA quantification was performed by the 2 −∆∆Ct method.Primer's sequences used in this paper are listed in Table 1.

RNase R digestion
Three μg of RNA was incubated with RNase R (Epicentre Biotechnologies, Madison, WI, USA) for 20 min under normal conditions.Expression of circCHSY1 and its linear gene CHSY1 was measured by qRT-PCR.

Transwell assay
Matrigel (Millipore, Billerica, MA, USA) was utilized for cell invasion analysis.In simple terms, 1 × 10 5 of ESCC cells with different substances (si-circCHSY1, si-NC, miRNA inhibitors, inhibitor control, miRNA mimics, mimic control, pcDNA-TCTN1, and pcDNA-NC) were added to the upper chambers.After 24 h of culture, the invaded cells were finally photographed under a microscope.

RNA pull-down assay
Eca109 and TE-1 cells were collected and incubated in a RIPA lysis buffer.The lysate was incubated with biotin-labeled oligonucleotide probes and Streptavidin-coupled Dynabeads (Invitrogen).The magnetic beads were incubated at 4 °C for 3 h and the combined RNA in the complex was purified using TRIzol.MiR-1224-3p, miR-1253, and miR-1229-3p expression were assessed by qRT-PCR.

Nucleus-cytoplasm fractionation
PARIS ™ kit (Invitrogen) was applied to determine the position of circCHSY1 in cells.The cytoplasmic and nuclear components of ESCC cells were isolated and collected for qRT-PCR analysis of circCHSY1 expression.

In vivo studies
Specific pathogen-free BALB/c nude female mice (n = 10) aged 4-5 weeks weighing 18-22 g were purchased from Hunan Slyke Jingda Experimental Animal Co., LTD (Changsha, China) and maintained in the absence of specific pathogens.The experiments were approved by the Administrative Panel on Laboratory Animal Care of Gaozhou People's Hospital.All animal procedures were carried out following the National guidelines of the Animal Care and Use of laboratory animals, the ARRIVE guidelines and the Basel Declaration.The maximal tumor size permitted by the Ethics Committee of Gaozhou People's Hospital is not exceed 2000 mm 3 in size and 20 mm in diameter.The generated xenograft tumors in this study were not exceeded this range.Two groups of mice were injected subcutaneously with TE-1 cells stably expressing sh-NC or sh-circCHSY1.The density of cell suspension was adjusted to 1 × 10 6 cells/mL, and each mouse was injected with the cell suspension (200 μL) to establish an in vivo model.Tumor volume was recorded weekly.After 4 weeks, the mice were anesthetized using pentobarbital sodium, and the tumor was dissected for tumor weight and gene expression analysis.Confounders were not controlled in this experiment.Immunohistochemistry (IHC) was conducted to analyze PCNA, Bax, Bcl-2, and Ki67 expression as instructed [18].Antibodies were obtained from Abcam (Shanghai) Trading Co., LTD.

Statistical analyses
Data in this study were analyzed by GraphPad Prism version 5.0 and presented as mean ± standard deviation.Distribution normality was evaluated using the Shapiro-Wilk normality test.The Student's t-test was used to analyze the difference between 2 groups.Analysis of variance was performed to analyze data among three or more groups.P-value less than 0.05 meant a significant difference.ESCC tissues and cells (Fig. 3E and F), so we speculated whether circCHSY1 could target to miR-1229-3p.The binding sites of circCHSY1 for miR-1229-3p are shown in Fig. 3G.The success of miR-1229-3p overexpression is presented in Fig. 3H.As expected, miR-1229-3p overexpression decreased the luciferase activity of circCHSY1 WT , which further proved the combination of the two RNAs (Fig. 3I and J).

MiR-1229-3p inhibitor restored tumorigenesis in circCHSY1-deficient cells
The data of CCK-8 (Fig. 4A and B) and EdU analysis (Fig. 4C) showed that circCHSY1 absence inhibited cell proliferation, but transfection of miR-1229-3p inhibitor reversed this inhibition.As shown in Fig. 4D, the cell apoptosis rate was significantly enhanced by decreasing the expression of circCHSY1, while anti-miR-1229-3p reduced the effect.At the protein level, the circCHSY1 deficiency increased Bax protein expression and decreased PCNA and Bcl-2 protein levels, while anti-miR-1229-3p attenuated these effects (Fig. 4E and F).In addition, knockdown circCHSY1 inhibited cell migration, invasion, and angiogenesis.However, these effects were mitigated by co-transfection with miR-1229-3p inhibitors (Fig. 4G-I).

Overexpression of TCTN1 alleviated the effects of miR-1229-3p in ESCC cells
The function of the miR-1229-3p/TCTN1 axis in ESCC cells was further explored through rescue experiments.The protein level of TCTN1 was significantly increased after transfection with TCTN1 overexpressed plasmid (Fig. 6A).CCK-8, EdU, and flow cytometry experiments showed that TCTN1 overexpression plasmid reversed the effects of miR-1229-3p on cell proliferation and apoptosis (Fig. 6B-G).In addition, miR-1229-3p resulted in significant suppression of migration, invasion, and angiogenesis in both ESCC cell lines, while overexpression of TCTN1 neutralized these effects (Fig. 6H-J).

CircCHSY1 targeted miR-1229-3p to regulate TCTN1
As shown in Fig. 7A and B, the deletion of circCHSY1 led to a decrease in TCTN1 expression, which was reversed by the miR-1229-3p inhibitor.

Knockdown of circCHSY1 hindered the growth of TE-1 cells in nude mice
Tumor size and tumor weight were suppressed by decreasing circCHSY1 expression (Fig. 8A-C).Besides, circCHSY1 and TCTN1 expression were decreased in the sh-circCHSY1 group (Fig. 8D and E).sh-circCHSY1 treatment decreased Ki-67, PCNA, and Bcl-2 levels and increased Bax level in tumors (Fig. 8F).

Discussion
CircRNAs can act as oncogenes or suppressor genes in various cancers by modulating signaling pathways [19].In ESCC, circ-SLC7A5 [20], circ-LRP6 [21] and circ-TTC17 [22] have been reported to play tumor-promoting roles, but circFoxo3 [23] and circSMAD7 [24] act tumor-inhibiting roles.In a previous study, researchers revealed that circCHSY1 was significantly overexpressed in ESCC [13].Although some evidence points to the involvement of circCHSY1 in disease progression through circ_0005019 [25], further research is required to gain a deeper understanding of the intricate networks regulated by circCHSY1.This might help us to develop clinical diagnostic reagents or seek more therapeutic targets.In this research, we discovered that circCHSY1 expression was increased in ESCC patients and cell lines.We observed that circCHSY1 silencing restrained cell migration, invasion, and angiogenesis, while also promoting apoptosis rate.Also, circCHSY1 silencing inhibited tumor growth in a xenograft mouse model using TE-1 cells.MiR-1229-3p is involved in the progression of many cancer tumors such as colorectal cancer, glioma, and hepatocellular carcinoma [26][27][28].In this study, we discovered this miRNA in tumor tissues and cells was significantly reduced, consistent with previous findings [29].Moreover, miR-1229-3p showed a significant negative correlation with circCHSY1.CircRNA functions include miRNA sponge, alternative splicing, and regulation of gene transcription [30,31].CircRNA indirectly affects gene expression through direct absorption of miRNA [32].Based on the above theories, we wondered whether circCHSY1 could sponge miR-1229-3p.Interestingly, in this project, miR-1229-3p inhibitor could reverse circCHSY1mediated changes in ESCC cells' function to a certain extent.
TCTN1 is a protein composed of 587 amino acids [33].It belongs to the tectonic family and is closely related to the Hedgehog signaling pathway [34].Early reports indicated that TCTN1 was associated with numerous tumors including gastric cancer [35], human thyroid cancer [36], as well as ESCC [37].In this topic, TCTN1 was abnormally elevated in ESCC tissues.TCTN1 was negatively modulated via miR-1229-3p and could participate in the changes in cell function caused by miR-1229-3p.Meanwhile, the absence of circCHSY1 reduced TCTN1 expression, whereas the inhibitor of miR-1229-3p reversed this phenomenon.These findings fully demonstrated that the novel signal axis circCHSY1/miR-1229-3p/TCTN1 was closely related to the regulation of ESCC.
However, there are some limitations to this research.Firstly, all clinical samples were collected from one hospital, and the sample size is relatively small.It is recommended to collect more samples from different hospitals in the future.In addition, the mechanism by which TCTN1 regulates ESCC progression has not been studied, and further experiments using other ESCC cell lines should be performed according to the methods described in this manuscript.The results show that the circCHSY1/miR-1229-3p/TCTN1 axis has a direct or indirect association with Bax, Bcl-2 and PCNA expression, and the relationship may have important influence on cell cycle regulation and apoptosis process.Further research is needed to test these hypotheses and explore their specific mechanisms of action.

Conclusion
In brief, this paper disclosed that circCHSY1, which was abnormally elevated in ESCC, could contribute to the malignant progression of ESCC.This effect was achieved through the regulation of circCHSY1 in the miR-1229-3p/TCTN1 pathway.More precisely, ESCC progression involved the increased circCHSY1 expression in ESCC cells, and circCHSY1 downregulation induced TCTN1 expression by segregating miR-1229-3p (Fig. 9).Accordingly, circCHSY1 could act as a promising therapeutic target in terms of ESCC.These results mean that we have a deeper understanding of how ESCC occurs, which could help develop more effective treatments.The development of new treatment strategies or drugs will provide more treatment options for patients with ESCC and help improve patient survival.

Fig. 1 Fig. 2
Fig. 1 CircCHSY1 was upregulated in ESCC tissues and cells.A The spliced mature sequence of circCHSY1 originated from the CHSY1 gene.B The expression of circCHSY1 in normal tissues (N = 53) and ESCC tissues (N = 53) was detected by qRT-PCR.C Expression of circCHSY1 in ESCC cell lines (TE-1, Eca-109, KYSE150 and KYSE79) was tested by qRT-PCR.D, E The expression of circCHSY1 and CHSY1 mRNA in ESCC cells was detected by qRT-PCR.The assay was performed with three independent biological replicates.*P < 0.05

Fig. 3 Fig. 4
Fig. 3 CircCHSY1 served as a sponge for miR-1229-3p.A, B The expression levels of GAPDH, U6 and circCHSY1 were determined by qRT-PCR in the cytoplasmic and nuclear parts of ESCC cells.C, D RNA pull-down assay was applied to identify the association of circCHSY1 with miR-1224-3p, miR-1253, and miR-1229-3p.E MiR-1229-3p expression in normal tissues (N = 53) and ESCC tissues (N = 53) was analyzed by qRT-PCR.F The level of miR-1229-3p in cells was detected with qRT-PCR.G The putative complementary sites of circCHSY1 and miR-1229-3p were predicted by bioinformatics analysis.H qRT-PCR was performed to analyze miR-1229-3p expression in ESCC cells transfected with miR-NC and miR-1229-3p.I, J Dual-luciferase reporter assays were implemented to analyze the interaction between circCHSY1 and miR-1229-3p.The assay was performed with three independent biological replicates.*P < 0.05

Fig. 5 Fig. 6
Fig. 5 MiR-1229-3p directly targeted TCTN1.A, B The mRNA and protein expression of TCTN1 in tissues was measured by qRT-PCR and western blot.C The expression of TCTN1 in ESCC cells and HET-1A cells was detected by western blot.D, E The linear associations between TCTN1 and miR-1229-3p and circCHSY1 were analyzed by Spearman's correlation coefficient.F The binding sites of miR-1229-3p and TCTN1 were predicted by the Targetscan.G, H The binding relationship was identified by a dual-luciferase reporter assay.I, J Western blot analysis was used to evaluate TCTN1 protein expression in TE-1 and Eca-109 cells after miR-1229-3p overexpression and knockdown.The assay was performed with three independent biological replicates.*P < 0.05

Fig. 9
Fig.9 The illustration showed the mechanism of circCHSY1 in regulating ESCC cell tumor properties.ESCC development involved circCHSY1 overexpression, and the increased expression of circCHSY1 induced TCTN1 production through miR-1229-3p, thereby promoting cell proliferation, migration, invasion, and angiogenesis and inhibiting cell apoptosis